Method for speeding up plant growth and improving yield by altering expression levels of kinases and phosphatases

ABSTRACT

Transgenic plants having increased growth rate and increase yield are disclosed, and methods for making the same. In one embodiment, the method comprises: transforming a plant or plant cell with a nucleic acid molecule comprising a plant kinase and/or phosphatase gene selected from NG6, NG21, NG24, NG28, and NG32, and over-expressing said kinase and/or phosphatase gene in the plant or plant cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/482,467, filed May 4, 2011, whichis hereby incorporated by reference in its entirety.

1. INTRODUCTION

Described herein are methods for speeding up plant growth and/orelevating plant yields by altering the expression levels of plantkinases and phosphatases. Also described therein are the use of plantkinases and phosphatases, and their respective protein products, as wellas fragments, derivatives, homologues, and variants thereof.

2. BACKGROUND OF THE INVENTION

Purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide rangeof activated phosphoric acid mono- and di-esters and anhydrides(Klabunde et al., 1996). The PAP proteins are characterized by sevenconserved amino acid residues (shown in bold face) in the five conservedmotifs XDXX, XDXXY, GNH(D/E), XXXH, XHXH, which are involved in thecoordination of the dimetal nuclear center (Fe³⁺-Me²⁺) in the activesite (Li et al., 2002), where Me is a transition metal; Me²⁺ is mostlyfound to be Fe²⁺ in mammals, and Zn²⁺, or Mn²⁺ in plants (Klabunde andKrebs, 1997; Schenk et al., 1999).

Multiple PAP-like sequences are present in plant genomes. In theArabidopsis genome, twenty-nine potential PAP genes have been identifiedbased on sequence comparison. Most of the functions of characterizedplant PAPs are related to phosphorus metabolism. None of the plant PAPsthat had been functionally or biochemically characterized carry anytransmembrane motif. In addition, no AtPAPs or any other plant PAPs hadbeen discovered to affect sugar signalling and carbon metabolism inplants. Overexpression of AtPAP2 in Arabidopsis, a PAP with a C-terminalmotif, can significantly speed up plant growth, increase sugar contentin plants and improve seed yield (U.S. Patent Application PublicationNo. 2010/0159065).

3. SUMMARY

In one aspect, provided herein are methods that speed up or increase therate of plant growth and elevate plant yields by altering the expressionlevels of plant kinases and phosphatases. Kinases and phosphatases, andtheir respectively encoded protein products, as well as fragments,derivatives, homologues, and variants thereof, are disclosed. Methodsfor introducing these genes into plants to speed up or increase thegrowth rate of plants, and to increase yield of plants, are provided.The kinases and phosphatases of the present invention are selected fromthe results of a microarray study. Surprisingly, it is discovered thatphosphatases (such as NG6) and kinases (such as NG21, NG24, NG28, andNG32) have growth-promoting effects.

Provided herein, a microarray study was carried out to compare the geneexpression profiles of the AtPAP2 overexpression lines, AtPAP2 T-DNA(mutant) line, and the wild-type plants. The results showed thatexpression levels of a number of genes are significantly altered(upregulated or downregulated) in AtPAP2 overexpression lines, whencompared to the wild-type. Among these genes, a number of phosphatasesand kinases were selected and analyzed using transgenic studies inArabidopsis.

At least in part, the present inventors discover that altering theexpression levels of plant phosphatases (such as NG6) and kinases (suchas NG21, NG24, NG28 and NG32) in plants resulted in rapid plant growthand higher yield. In one aspect, provided herein are methods ofproducing plants with enhanced growth and/or yield. In one embodiment,the method comprises: transforming a plant or plant cell with a nucleicacid molecule comprising a plant kinase and/or phosphatase gene selectedfrom SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99, or 101, andover-expressing said kinase and/or phosphatase gene in the plant orplant cell. In one embodiment, provided herein are methods ofregenerating, from said transformed plant or plant cell, a plant havingenhanced growth and/or yield.

In one embodiment, the method comprises: transforming a plant or plantcell with a nucleic acid molecule comprising a plant kinase and/orphosphatase having at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%,97%, 98%, or 99% identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95,97, 99, or 101, and over-expressing said kinase and/or phosphatase genein the plant or plant cell.

In certain embodiments, the method comprises transforming a plant orplant cell with a nucleic acid molecule comprising a plant kinase and/orphosphatase having a nucleic acid fragment from SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 93, 95, 97, 99 or 101. In certain embodiments, the nucleicacid fragment encode a peptide that has the same activity as a peptideencoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99 or 101.

In certain embodiments, the activity is a kinase and/or phosphataseactivity. In certain, embodiments, the method comprises transforming aplant or plant cell with a nucleic and molecule comprising a plantkinase and/or phosphatase having a variant from SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 93, 95, 97, 99 or 101.

In certain embodiments, the variant has 1-5, 6-10, 11-20, 21-30, 31-40,41-50, 50-70, 71-80, 81-100 nucleic acid deletion, substitution orinsertion in the sequence as compared to SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 93, 95, 97, 99, or 101. In certain embodiments, the variants encodea peptide that has the same activity as a peptide encoded by SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 93, 95, 97, 99, or 101. In certain embodiments,the activity is a kinase and/or phosphatase activity.

Provided herein are transgenic plants with enhanced growth and/or yield.In certain embodiments, the transgenic plant comprises a nucleic acidmolecule selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99,or 101, wherein said nucleic acid molecule is overexpressed in thetransgenic plant when compared to a wild-type plant of the same speciescultivated under the same conditions.

In certain embodiments, the transgenic plant comprises a nucleic acidmolecule having at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%,97%, 98% or 99% identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95,97, 99, or 101, wherein said nucleic acid molecule is overexpressed inthe transgenic plant when compared to a wild-type plant of the samespecies cultivated under the same conditions.

In certain embodiments, the transgenic plant comprises a nucleic acidfragment from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99, or 101.In certain embodiments, the nucleic acid fragment encodes a peptide thathas the same activity as a peptide encoded by SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 93, 95, 97, 99, or 101. In certain embodiments, the activity isa kinase and/or phosphatase activity.

In certain embodiments, the transgenic plant comprises a plant kinaseand/or phosphatase homologue, derivative, or variant having a nucleicacid sequence of the SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99,or 101. In certain embodiments, the homologue, derivative or variant has1-5, 6-10, 11-20, 21-30, 31-40, 41-50, 50-70, 71-80, 81-100 nucleic aciddeletion, substitution or insertion in the sequence as compared to SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99, or 101. In certainembodiments, the variants encode a peptide that has the same activity asa peptide encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99or 101. In certain embodiments, the activity is a kinase and/orphosphatase activity.

In certain embodiments provided herein are the methods of altering theexpression levels of plant kinase and/or phosphatase. In certainembodiments, the method comprises transforming a plant or plant cellwith a nucleic acid molecule that expresses a plant kinase and/orphosphatase peptide, fragment, derivative or variant from a peptidehaving an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94,96, 98, 100, or 102. In certain embodiments, the peptide, fragment,derivative or variant is overexpressed. In certain embodiments, providedherein are methods of regenerating, from said transformed plant or plantcell, a plant having enhanced growth and/or yield.

In certain embodiments, the transgenic plants express a plant kinaseand/or phosphatase peptide, fragment, derivative or variant from aprotein having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 94, 96, 98, 100, or 102. In certain embodiments, the peptidefragment, derivative or variant is overexpressed. In certainembodiments, provided herein are regenerated transformed plant havingenhanced growth and/or yield.

4. BRIEF DESCRIPTION OF THE FIGURES

The patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a heat map of the microarray analysis of gene expressionprofile of Arabidopsis shoots, using three biological replicates forwild-type (WT), 2 biological replicates for AtPAP2 T-DNA line (P2), and3 biological replicates for two independent AtPAP2 overexpression lines(OE7 and OE21).

FIG. 2 shows scatter plots of the microarray analysis of gene expressionprofile of Arabidopsis shoots. The results showed that the expressionprofiles of the two independent AtPAP2 overexpression lines (OE7 andOE21) were significantly different from that of the wild-type (WT),whereas the expression profile of the AtPAP2 T-DNA (mutant) linesresembled closely that of the WT.

FIG. 3 shows a schematic diagram of the expression vector pCXSN. (a).The cDNAs of the NG genes were cloned into the pCXSN vector at the XcmIsites to create the overexpression vectors. (b) shows an exemplifiedoverexpression vector pCXSN-NG6.

FIG. 4 shows the mRNA expression levels of NG genes in the respectiveoverexpression lines. The mRNA expression levels in 10-day-old T3homologous seedlings were determined by quantitative RT-PCR usinggene-specific primers. The fold-changes represent the relativeexpression levels of mRNAs compared to that of the wild-type (WT=1.0).The results of two trials were obtained from two batches of plant growthstudies.

FIG. 5 shows the growth performance of the wild-type and NG6over-expression lines in soil. The five columns of plants from left toright were AtPAP2 overexpression lines, WT, T3 homologous NG6overexpression lines NG6-1, NG6-2, and NG6-3. (a) 22-day-old and (b)25-day-old plants.

FIG. 6 shows the growth performance of the wild-type and T3 homologousNG21, NG24, NG28 and NG32 overexpression lines in soil. The five columnsof plants from left to right were WT, NG21, NG24, NG28 and NG32overexpression lines. (a) 30-day-old plants and (b) 34-day-old plantsgrown in black tray. (c) 22-day-old plants, (d) 25-day-old plants, (e)28-day-old plants, and (f) 36-day-old plants grown in white cups.

4.1. BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleic acid sequence of Arabidopsis phosphatase NG6gene.

SEQ ID NO:2 is an amino acid sequence of Arabidopsis phosphatase NG6.

SEQ ID NO:3 is a nucleic acid sequence of maize phosphatase NG6 gene.

SEQ ID NO:4 is an amino acid sequence of maize phosphatase NG6.

SEQ ID NO:5 is a nucleic acid sequence of soybean phosphatase NG6 gene.

SEQ ID NO:6 is an amino acid sequence of soybean phosphatase NG6.

SEQ ID NO:7 is a nucleic acid sequence of rice phosphatase NG6 gene.

SEQ ID NO:8 is an amino acid sequence of rice phosphatase NG6.

SEQ ID NO:9 is a nucleic acid sequence of cotton phosphatase NG6 gene.

SEQ ID NO:10 is an amino acid sequence of cotton phosphatase NG6.

SEQ ID NO:11 is a nucleic acid sequence of Arabidopsis kinase NG21 gene.

SEQ ID NO:12 is an amino acid sequence of Arabidopsis kinase NG21.

SEQ ID NO:13 is a nucleic acid sequence of maize kinase NG21 gene.

SEQ ID NO:14 is an amino acid sequence of maize kinase NG21.

SEQ ID NO:15 is a nucleic acid sequence of soybean kinase NG21 gene.

SEQ ID NO:16 is an amino acid sequence of soybean kinase NG21.

SEQ ID NO:17 is a nucleic acid sequence of rice kinase NG21 gene.

SEQ ID NO:18 is an amino acid sequence of rice kinase NG21.

SEQ ID NO:19 is a nucleic acid sequence of cotton kinase NG21 gene.

SEQ ID NO:20 is an amino acid sequence of cotton kinase NG21.

SEQ ID NO:21 is a nucleic acid sequence of Arabidopsis kinase NG24 gene.

SEQ ID NO:22 is an amino acid sequence of Arabidopsis kinase NG24.

SEQ ID NO:23 is a nucleic acid sequence of maize kinase NG24 gene.

SEQ ID NO:24 is an amino acid sequence of maize kinase NG24.

SEQ ID NO:25 is a nucleic acid sequence of soybean kinase NG24 gene.

SEQ ID NO:26 is an amino acid sequence of soybean kinase NG24.

SEQ ID NO:27 is a nucleic acid sequence of rice kinase NG24 gene.

SEQ ID NO:28 is an amino acid sequence of rice kinase NG24.

SEQ ID NO:29 is a nucleic acid sequence of cotton kinase NG24 gene.

SEQ ID NO:30 is an amino acid sequence of cotton kinase NG24.

SEQ ID NO:31 is a nucleic acid sequence of Arabidopsis kinase NG28 gene.

SEQ ID NO:32 is an amino acid sequence of Arabidopsis kinase NG28.

SEQ ID NO:33 is a nucleic acid sequence of maize kinase NG28 gene.

SEQ ID NO:34 is an amino acid sequence of maize kinase NG28.

SEQ ID NO:35 is a nucleic acid sequence of soybean kinase NG28 gene.

SEQ ID NO:36 is an amino acid sequence of soybean kinase NG28.

SEQ ID NO:37 is a nucleic acid sequence of rice kinase NG28 gene.

SEQ ID NO:38 is an amino acid sequence of rice kinase NG28.

SEQ ID NO:39 is a nucleic acid sequence of cotton kinase NG28 gene.

SEQ ID NO:40 is an amino acid sequence of cotton kinase NG28.

SEQ ID NO:41 is a nucleic acid sequence of Arabidopsis kinase NG32 gene.

SEQ ID NO:42 is an amino acid sequence of Arabidopsis kinase NG32.

SEQ ID NO:43 is a nucleic acid sequence of maize kinase NG32 gene.

SEQ ID NO:44 is an amino acid sequence of maize kinase NG32.

SEQ ID NO:45 is a nucleic acid sequence of soybean kinase NG32 gene.

SEQ ID NO:46 is an amino acid sequence of soybean kinase NG32.

SEQ ID NO:47 is a nucleic acid sequence of rice kinase NG32 gene.

SEQ ID NO:48 is an amino acid sequence of rice kinase NG32.

SEQ ID NO:49 is a nucleic acid sequence of cotton kinase NG32 gene.

SEQ ID NO:50 is an amino acid sequence of cotton kinase NG32.

SEQ ID NO:51 is a primer sequence useful according to the presentinvention.

SEQ ID NO:52 is a primer sequence useful according to the presentinvention.

SEQ ID NO:53 is a primer sequence useful according to the presentinvention.

SEQ ID NO:54 is a primer sequence useful according to the presentinvention.

SEQ ID NO:55 is a primer sequence useful according to the presentinvention.

SEQ ID NO:56 is a primer sequence useful according to the presentinvention.

SEQ ID NO:57 is a primer sequence useful according to the presentinvention.

SEQ ID NO:58 is a primer sequence useful according to the presentinvention.

SEQ ID NO:59 is a primer sequence useful according to the presentinvention.

SEQ ID NO:60 is a primer sequence useful according to the presentinvention.

SEQ ID NO:61 is a primer sequence useful according to the presentinvention.

SEQ ID NO:62 is a primer sequence useful according to the presentinvention.

SEQ ID NO:63 is a primer sequence useful according to the presentinvention.

SEQ ID NO:64 is a primer sequence useful according to the presentinvention.

SEQ ID NO:65 is a primer sequence useful according to the presentinvention.

SEQ ID NO:66 is a primer sequence useful according to the presentinvention.

SEQ ID NO:67 is a primer sequence useful according to the presentinvention.

SEQ ID NO:68 is a primer sequence useful according to the presentinvention.

SEQ ID NO:69 is a primer sequence useful according to the presentinvention.

SEQ ID NO:70 is a primer sequence useful according to the presentinvention.

SEQ ID NO:71 is a primer sequence useful according to the presentinvention.

SEQ ID NO:72 is a primer sequence useful according to the presentinvention.

SEQ ID NO:73 is a nucleic acid sequence of Arabidopsis AtPAP2phosphatase gene.

SEQ ID NO:74 is an amino acid sequence of Arabidopsis AtPAP2phosphatase.

SEQ ID NO:75 is an amino acid sequence of a conserved motif of an NG6protein.

SEQ ID NO:76 is an amino acid sequence of a conserved motif of an NG6protein.

SEQ ID NO:77 is an amino acid sequence of a conserved motif of an NG6protein.

SEQ ID NO:78 is an amino acid sequence of a conserved motif of an NG6protein.

SEQ ID NO:79 is an amino acid sequence of a conserved motif of an NG21protein.

SEQ ID NO:80 is an amino acid sequence of a conserved motif of an NG21protein.

SEQ ID NO:81 is an amino acid sequence of a conserved motif of an NG21protein.

SEQ ID NO:82 is an amino acid sequence of a conserved motif of an NG21protein.

SEQ ID NO:83 is an amino acid sequence of a conserved motif of an NG24protein.

SEQ ID NO:84 is an amino acid sequence of a conserved motif of an NG24protein.

SEQ ID NO:85 is an amino acid sequence of a conserved motif of an NG24protein.

SEQ ID NO:86 is an amino acid sequence of a conserved motif of an NG28protein.

SEQ ID NO:87 is an amino acid sequence of a conserved motif of an NG28protein.

SEQ ID NO:88 is an amino acid sequence of a conserved motif of an NG28protein.

SEQ ID NO:89 is an amino acid sequence of a conserved motif of an NG32protein.

SEQ ID NO:90 is an amino acid sequence of a conserved motif of an NG32protein.

SEQ ID NO:91 is an amino acid sequence of a conserved motif of an NG32protein.

SEQ ID NO:92 is an amino acid sequence of a conserved motif of an NG32protein.

SEQ ID NO:93 is a nucleic acid sequence of rapeseed kinase NG6 gene.

SEQ ID NO:94 is an amino acid sequence of rapeseed kinase NG6.

SEQ ID NO:95 is a nucleic acid sequence of rapeseed kinase NG21 gene.

SEQ ID NO:96 is an amino acid sequence of rapeseed kinase NG21.

SEQ ID NO:97 is a nucleic acid sequence of rapeseed kinase NG24 gene.

SEQ ID NO:98 is an amino acid sequence of rapeseed kinase NG24.

SEQ ID NO:99 is a nucleic acid sequence of rapeseed kinase NG28 gene.

SEQ ID NO:100 is an amino acid sequence of rapeseed kinase NG28.

SEQ ID NO:101 is a nucleic acid sequence of rapeseed kinase NG32 gene.

SEQ ID NO:102 is an amino acid sequence of rapeseed kinase NG32.

5. DETAILED DESCRIPTION

Provided herein are methods of producing plants with enhanced growthand/or yield. In one embodiment, the method comprises: transforming aplant or plant cell with a nucleic acid molecule comprising a plantkinase and/or phosphatase gene selected from NG6, NG21, NG24, NG28, andNG32, and over-expressing said kinase and/or phosphatase gene in theplant or plant cell. In one embodiment, the method further comprises:regenerating, from said transformed plant or plant cell, a plant havingenhanced growth and/or yield. Also provided are transgenic plants withenhanced growth and/or yield, comprising a plant kinase and/orphosphatase gene selected from NG6, NG21, NG24, NG28, and NG32, whereinthe kinase and/or phosphatase is overexpressed in the plant or plantcell.

The inventors discover that altering the expression levels of one ormore phosphatases (such as NG6) and kinases (such as NG21, NG24, NG28,and NG32) results in rapid plant growth and higher yield. The geneexpression profiles of the AtPAP2 overexpression lines, AtPAP2 T-DNA(mutant) line, and the wild-type plants are analyzed using microarray.The microarray data show that the expression levels of a range of genesare significantly altered (upregulated or downregulated) in the AtPAP2overexpression lines, when compared to the wild-type.

The introduction of a representative gene of phosphatases (AT1G05000(NG6)) and kinases (AT1G13350 (NG21), AT1G28390 (NG24), AT3G24660 (NG28)and AT5G03320 (NG32)), into the genome of Arabidopsis by transgenictechnology produced transgenic Arabidopsis that grew faster than thewild-type plants (Table 4, FIG. 5, FIG. 6), and the yield of seeds wereelevated by 23-70% (Table 5).

While any plant species can be modified using the methods describedherein, preferably included without limitation are species from thefollowing genera with representative species in parentheses:

Monocots: genera Asparagus (asparagus), Bromus (cheatgrass),Hemerocallis (daylily), Hordeum (barley), Lolium (ryegrass), Oryza(rice), Panicum (Switchgrass), Pennisetum (fountaingrass), Saccharum(Sugar cane), Sorghum, Trigonella (fenu grass), Triticum (wheat), andZea (corn); and

Dicots: genera Antirrhinum (flower sp.), Arabidopsis (thaliana), Arachis(peanut), Atropa (deadly nightshade), Brassica (rapeseed), Browallia,Capsicum (pepper), Carthamus (safflower), Cichorium (chicory), Citrus(orange, lemon), Chrysanthemum, Cucumis (cucumber), Datura (thornapple), Daucus (carrot), Digitalis (foxglove), Fragaria (strawberry),Geranium (flower sp.), Glycine (soybean), Helianthus (sunflower),Hyscyamus, Ipomoea (morning glory), Latuca (lettuce), Linum (linseed),Lotus (flower sp.), Lycopersicon (tomato), Majorana, Malva (cotton),Manihot, Medicago (alfalfa), Nemesia, Nicotiana (tobacco), Onobrychis,Pelargonium (citrosa), Petunia (flower sp.), Ranunculus (flower sp.),Raphanus (radishes), Salpiglossis, Senecio (flower sp.), Sinapis (albaesemen), Solanum (potato), Trifolium (clovers), Vigna (mungbean, favabean), and Vitis (grape).

In certain embodiments, plant species transgenically modified accordingto the present invention are selected from soybean, maize, potato, rice,sugar canes, switchgrass, cotton, sorghum, alfalfas, rapeseed, canola,rye, sorghum, sunflower, wheat, tobacco, millet, peanuts sweet potatocassava, coffee, coconut, cocoa, tea, banana, citrus, apple, pineapple,avocado, fig, guava, mango, olive, barley ornamentals, and conifers. Inpreferred embodiments, plant species transgenically modified accordingto the present invention are selected from soybean, maize, potato, rice,sugar canes, switchgrass, cotton, sorghum, alfalfas, rapeseed, andcanola.

In certain embodiment, plant parts, plant tissue, and plant cellsincluding, but not limited to, shoots, stems, seeds, and roots, can betransgenically modified in accordance with the present invention.

4.2 Definitions

The term “protein or peptide homologue,” as used herein, refers to oneor more of the following proteins or peptides: (i) a protein orpolypeptide with at least about 60%, at least about 70%, at least about80%, at least about 90%, or at least about 98% sequence identity with aprotein or polypeptide of the invention; (ii) a protein or polypeptideencoded by a nucleotide sequence that is at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or at least about 98%identical to a nucleic acid sequence of the invention; (iii) a proteinor polypeptide encoded by a nucleotide sequence that hybridizes understringent conditions to a nucleotide sequence of the invention; (iv) aprotein or polypeptide that is derived from conservative substitution ofamino acids of a protein or polypeptide of the invention, or that isderived from conservative substitution of amino acids of a protein orpolypeptide of (i)-(iii); (v) a fragment of a protein or polypeptide ofthe invention or a fragment of a protein or polypeptide of (i) through(iv); and (vii) a protein or polypeptide recognized by an antibody thatimmunospecifically binds to a sequence selected from SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 94, 96, 98, 100 or 102.

The term “an antibody or an antibody fragment that immunospecificallybinds to a polypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,94, 96, 98, 100 or 102” or “an antibody or an antibody fragment thatimmunospecifically binds to a polypeptide, peptide, or protein of theinvention,” as used herein, refers to an antibody or a fragment thereofthat immunospecifically binds to a polypeptide selected from SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 94, 96, 98, 100 or 102, or a fragment of thesepolypeptide, wherein the antibody or the antibody fragment does notnon-specifically bind to other peptides, polypeptides, or proteins.

An antibody or a fragment thereof that immunospecifically binds to apolypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98,100 or 102, or a fragment of these polypeptide, may cross-react withother antigens. In a preferred embodiment, an antibody or a fragmentthereof that immunospecifically binds to a polypeptide selected from SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98, 100 or 102, or a fragment ofthese polypeptides, does not cross-react with other antigens. Anantibody or a fragment thereof that immunospecifically binds to apolypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98,100 or 102, or a fragment of these polypeptide, can be identified by,for example, immunoassays or other techniques known to those skilled inthe art. An antibody or an antibody fragment that immunospecificallybinds a polypeptide selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94,96, 98, 100 or 102 may be interchangeably referred to as “anti-PAPantibody”.

The term “peptide or protein derivative,” as used herein, refers to agiven peptide or protein that is modified, e.g., by covalent attachmentof another molecule, to the peptide or protein, including theincorporation of non-naturally occurring amino acids. The peptide orprotein derivative retains one or more biological activities of thepeptide or protein.

The term “nucleic acid fragment,” as used herein, refers to a fragmentof a nucleic acid molecule of the invention, wherein the fragmentcomprises at least about 400, at least about 450, at least about 500, atleast about 550, at least about 600, at least about 650, at least about700, at least about 750, at least about 800, at least about 850, atleast about 900, at least about 950, at least about 1000, at least about1050, at least about 1100, at least about 1150, at least about 1200, atleast about 1250, at least about 1300, or at least about 1350 contiguousnucleic acid bases of the nucleic acid molecule.

The term “protein or peptide fragment,” as used herein, refers to afragment of a protein or peptide of the invention, wherein the fragmentcomprises at least about 160, at least about 180, at least about 200, atleast about 220, at least about 240, at least about 260, at least about280, at least about 300, at least about 320, at least about 340, or atleast about 360 contiguous amino acid residues of the protein orpeptide.

The term “protein or peptide variant,” as used herein, includes 1) anaturally occurring allelic variation of a given protein or peptide, and2) a recombinantly prepared variation of a given protein or peptide, inwhich one or more amino acid residues have been modified by amino acidsubstitution, addition, and/or deletion.

An “isolated” nucleic acid molecule has been removed from anyenvironment in which it may exist in nature. For instance, an “isolated”nucleic acid molecule, such as a cDNA molecule, is substantially free ofother cellular materials, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. In a preferred embodiment,nucleic acid molecules encoding the polypeptides/proteins of the presentinvention are isolated or purified.

The term “under stringent conditions” refers to hybridization andwashing conditions under which nucleotide sequences having homology toeach other remain hybridized to each other. Such hybridizationconditions are described in, for example, but not limited to, CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6.; Basic Methods in Molecular Biology, Elsevier SciencePublishing Co., Inc., N.Y. (1986), pp. 75-78, and 84-87; and MolecularCloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp. 387-389, andare well known to those skilled in the art. A preferred example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC), 0.5% SDS at about 68° C. followed by oneor more washes in 2×SSC, 0.5% SDS at room temperature. Anotherpreferred, example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C. followed by one or more washes in0.2×SSC, 0.1% SDS at about 50-65° C.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. U.S.A. 87:2264 2268, modified as in Karlin and Altschul,1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm isincorporated into the NBLAST and) (BLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the present invention. BLAST protein searches can beperformed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule of the present invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g.,the NCBI website). Another preferred, non limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithmis incorporated in the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the term “derivative” (e.g., proteins, polypeptides,peptides, and antibodies) refers to an agent that comprises an aminoacid sequence which has been altered by the introduction of amino acidresidue substitutions, deletions, and/or additions. The term“derivative” as used herein also refers to an agent which has beenmodified, i.e., by the covalent attachment of any type of molecule tothe agent. For example, but not by way of limitation, an antibody may bemodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. A derivative of an agent may be produced by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Further, aderivative of an agent may contain one or more non-classical aminoacids. A derivative of an agent possesses a similar or identicalfunction as the agent from which it was derived.

The term “enhance or promote plant growth and/or yield” refers to forexample, increased plant weight, increased leaf number and/or weight,increased number of inflorescence, increased seed production (such asweight/seed and total weight of seeds), increased carbon metabolism,increased carbohydrate (e.g., starch, sugars, cellulose), amino acid,and/or lipid production, early bolting, and also can includecombinations of the foregoing, when compared to a wild-type plant of thesame species cultivated under the same conditions.

5.1 Growth-promoting Phosphatases and Kinases

Provided herein are phosphatases and kinases that promote plant growthand/or yield. In one embodiment, the growth-promoting phosphatase isNG6, and the growth-promoting kinases are selected from NG6, NG21, NG24,NG28, and NG32. In certain specific embodiments, the growth-promotingphosphatases and kinases are derived from plant species including, butnot limited to, Arabidopsis, rice, soybean, maize, and cotton.

In certain embodiments, the phosphatase gene that promotes plant growthand/or yield is an NG6 gene comprising a nucleic acid sequence selectedfrom SEQ ID NO: 1, 3, 5, 7, 9 or 93. In certain embodiments, thephosphatase gene that promotes plant growth and/or yield comprises anucleic acid sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%,98% or 99% identity with SEQ ID NO: 1, 3, 5, 7, 9 or 93. In certainembodiments, the phosphatase gene that promotes plant growth and/oryield is a homologue, derivative, or variant of a nucleic acid moleculederives from a nucleic acid molecule having nucleic acid sequencecomprising SEQ ID NO: 1, 3, 5, 7, 9 or 93.

In certain embodiments, the phosphatase gene that promotes plant growthand/or yield comprises a nucleic acid sequence having at least 80%, 85%,90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1, 3, 5, 7,9 or 93. In certain embodiments, the phosphatase gene that promotesplant growth and/or yield comprises the nucleic acid sequence thatencodes a protein that comprises one or more of the following conservedmotifs: GIFRSGFP (SEQ ID NO:75), YLCPEPYP (SEQ ID NO:76), KEPFVXIP (SEQID NO:77), and HCXRGKHRTG (SEQ ID NO:78).

In certain embodiments, the phosphatase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequences comprising SEQ ID NO: 1, 3, 5, 7, 9 or 93. In certainembodiments, the phosphatase gene that promotes plant growth and/oryield comprises one or more of the following conserved motifs: GIFRSGFP(SEQ ID NO:75), YLCPEPYP (SEQ ID NO:76), KEPFVXIP (SEQ ID NO:77), andHCXRGKHRTG (SEQ ID NO:78).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is an NG21 gene comprising a nucleic acid sequence selectedfrom SEQ ID NO: 11, 13, 15, 17, 19 or 95. In certain embodiments, thekinase gene that promotes plant growth and/or yield comprises a nucleicacid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%,96%, 97%, 98% or 99% identity with SEQ ID NO: 11, 13, 15, 17, 19 or 95.In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequence comprising SEQ ID NO: 11, 13, 15, 17, 19 or 95.

In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises a nucleic acid sequence having at least 80%, 85%,90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 11, 13, 15,17, or 19 or 95. In certain embodiments, the kinase gene that promotesplant growth and/or yield comprises the nucleic acid sequence thatencodes a protein that comprises one or more of the following conservedmotifs: DNWDDA(D/E)GYY (SEQ ID NO:79), YRNHLCLVFESL (SEQ ID NO:80),VLHCDIKPDNMLVNE (SEQ ID NO:81), and TPYLVSRFYRXPEI (SEQ ID NO:82).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequences comprising SEQ ID NO: 11, 13, 15, 17, 19 or 95. In ceratinembodiments, the kinase gene that promotes plant growth and/or yieldcomprises one or more of the following conserved motifs: DNWDDA(D/E)GYY(SEQ ID NO:79), YRNHLCLVFESL(SEQ ID NO:80), VLHCDIKPDNMLVNE (SEQ IDNO:81), and TPYLVSRFYRXPEI (SEQ ID NO:82). In certain embodiments, thekinase gene that promotes plant growth and/or yield is an NG24 genecomprising a nucleic acid sequence selected from SEQ ID NO: 21, 23, 25,27, 29 or 97. In certain embodiments, the kinase gene that promotesplant growth and/or yield comprises a nucleic acid sequence having atleast 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99%identity with SEQ ID NO: 21, 23, 25, 27, 29 or 97. In certainembodiments, the kinase gene that promotes plant growth and/or yield isa homologue, derivative, or variant of a nucleic acid molecule thatderives from nucleic acid molecule having nucleic acid sequencecomprising SEQ ID NO: 21, 23, 25, 27, 29 or 97.

In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises a nucleic acid sequence having at least 80%, 85%,90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 21, 23, 25,27, 29 or 97. In certain embodiments, the kinase gene that promotesplant growth and/or yield comprises one or more of the followingconserved motifs: VRHRDXKS (SEQ ID NO:83), GTLXGYLDP (SEQ ID NO:84), andDV(F/Y)S(F/Y)G(I/V)LLLEI (SEQ ID NO:85).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequence comprising SEQ ID NO: 21, 23, 25, 27, 29 or 97. In certainembodiments, the kinase gene that promotes plant growth and/or yieldcomprises one or more of the following conserved motifs: VRHRDXKS (SEQID NO:83), GTLXGYLDP (SEQ ID NO:84), and DV(F/Y)S(F/Y)G(I/V)LLLEI (SEQID NO:85). In certain embodiments, the kinase gene that promotes plantgrowth and/or yield is an NG28 gene comprising a nucleic acid sequenceselected from SEQ ID NO: 31, 33, 35, 37, 39 or 99. In certainembodiments, the NG24 kinase gene that promotes plant growth and/oryield comprises a nucleic acid sequence having at least 65%, 70%, 75%,80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:31, 33, 35, 37, 39 or 99. In certain embodiments, the NG24 kinase genethat promotes plant growth and/or yield is a homologue, derivative, orvariant of a nucleic acid molecule that derives from nucleic acidmolecule having nucleic acid sequence comprising SEQ ID NO: 31, 33, 35,37, 39 or 99.

In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises a nucleic acid sequence having at least 80%, 85%,90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 31, 33, 35,37, 39 or 99. In certain embodiments, the kinase gene that promotesplant growth and/or yield comprises one or more of the followingconserved motifs: RRHKIALG (SEQ ID NO:86), Y(K/R)APEL (SEQ ID NO:87),and DVYAFGILLLE (SEQ ID NO:88).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequence comprising SEQ ID NO: 31, 33, 35, 37, 39 or 99. In certainembodiments, the kinase gene that promotes plant growth and/or yieldcomprises one or more of the following conserved motifs: RRHKIALG (SEQID NO:86), Y(K/R)APEL (SEQ ID NO:87), and DVYAFGILLLE (SEQ ID NO:88).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is an NG32 gene comprising a nucleic acid sequence selectedfrom SEQ ID NO: 41, 43, 45, 47, 49 or 101. In certain embodiments, thekinase gene that promotes plant growth and/or yield comprises a nucleicacid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%,96%, 97%, 98% or 99% identity with SEQ ID NO: 41, 43, 45, 47, 49 or 101.In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequence comprising SEQ ID NO: 41, 43, 45, 47, 49 or 101.

In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises a nucleic acid sequence having at least 80%, 85%,90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 41, 43, 45,47, 49 or 101. In certain embodiments, the kinase gene that promotesplant growth and/or yield comprises one or more of the followingconserved motifs: CAXDDERG (SEQ ID NO:89), AKLSDFGLAR (SEQ ID NO:90),YELITGR(R/K) (SEQ ID NO:91), and RPKMSEV (SEQ ID NO:92).

In certain embodiments, the kinase gene that promotes plant growthand/or yield is a homologue, derivative, or variant of a nucleic acidmolecule that derives from nucleic acid molecule having nucleic acidsequence comprising SEQ ID NO: 41, 43, 45, 47, 49 or 101. In certainembodiments, the kinase gene that promotes plant growth and/or yieldcomprises one or more of the following conserved motifs: CAXDDERG (SEQID NO:89), AKLSDFGLAR (SEQ ID NO:90), YELITGR(R/K) (SEQ ID NO:91), andRPKMSEV (SEQ ID NO:92). In certain embodiments, the phosphatase orkinase gene that promotes plant growth and/or yield encodes a proteinselected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98, 100 or 102.In certain embodiments, the phosphatase or kinase gene that promotesplant growth and/or yield encodes a protein having at least 65%, 70%,75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 94, 96, 98, 100 or 102. In certainembodiments, the phosphatase or kinase gene that promotes plant growthand/or yield encodes a protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94,96, 98, 100 or 102.

In certain embodiments, the phosphatase gene that promotes plant growthand/or yield encodes an NG6 protein having at least 80%, 85%, 90%, 93%,95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2, 4, 6, 8, 10 or 94.In certain embodiments, the phosphatase gene that promotes plant growthand/or yield one or more of the following conserved motifs: GIFRSGFP(SEQ ID NO:75), YLCPEPYP (SEQ ID NO:76), KEPFVXIP (SEQ ID NO:77), andHCXRGKHRTG (SEQ ID NO:78).

In certain embodiments, the phosphatase gene that promotes plant growthand/or yield encodes an NG6 protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence comprising SEQ ID NO: 2, 4, 6, 8, 10 or 94. Incertain embodiments, the phosphatase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:GIFRSGFP (SEQ ID NO:75), YLCPEPYP (SEQ ID NO:76), KEPFVXIP (SEQ IDNO:77), and HCXRGKHRTG (SEQ ID NO:78).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG 21 protein having at least 80%, 85%, 90%,93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 12, 14, 16, 18,20 or 96. In certain embodiments, the kinase gene that promotes plantgrowth and/or yield comprises one or more of the following conservedmotifs: DNWDDA(D/E)GYY (SEQ ID NO:79), YRNHLCLVFESL (SEQ ID NO:80),VLHCDIKPDNMLVNE (SEQ ID NO:81), and TPYLVSRFYRXPEI (SEQ ID NO:82).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG21 protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence selected from SEQ ID NO: 12, 14, 16, 18, 20 or 96.In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:DNWDDA(D/E)GYY (SEQ ID NO:79), YRNHLCLVFESL (SEQ ID NO:80),VLHCDIKPDNMLVNE (SEQ ID NO:81), and TPYLVSRFYRXPEI (SEQ ID NO:82).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG24 protein having at least 80%, 85%, 90%, 93%,95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 22, 24, 26, 28, 30 or98. In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:VRHRDXKS (SEQ ID NO:83), GTLXGYLDP (SEQ ID NO:84), andDV(F/Y)S(F/Y)G(I/V)LLLEI (SEQ ID NO:85).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG24 protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence selected from SEQ ID NO: 22, 24, 26, 28, 30 or 98.In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:VRHRDXKS (SEQ ID NO:83), GTLXGYLDP (SEQ ID NO:84), andDV(F/Y)S(F/Y)G(UV)LLLEI (SEQ ID NO:85).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG28 protein having at least 80%, 85%, 90%, 93%,95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 32, 34, 36, 38, 40 or100. In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:RRHKIALG (SEQ ID NO:86), Y(K/R)APEL (SEQ ID NO:87), and DVYAFGILLLE (SEQID NO:88).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG28 protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence selected from SEQ ID NO: 32, 34, 36, 38, 40 or 100,wherein the protein comprises one or more of the following conservedmotifs: RRHKIALG (SEQ ID NO:86), Y(K/R)APEL (SEQ ID NO:87), andDVYAFGILLLE (SEQ ID NO:88).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG32 protein having at least 80%, 85%, 90%, 93%,95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 42, 44, 46, 48, 50 or102. In certain embodiments, the kinase gene that promotes plant growthand/or yield comprises one or more of the following conserved motifs:CAXDDERG (SEQ ID NO:89), AKLSDFGLAR (SEQ ID NO:90), YELITGR(R/K) (SEQ IDNO:91), and RPKMSEV (SEQ ID NO:92).

In certain embodiments, the kinase gene that promotes plant growthand/or yield encodes an NG32 protein that is a homologue, derivative, orvariant of a protein derived from the amino acid molecule having theamino acid sequence selected from SEQ ID NO: 42, 44, 46, 48, 50 or 102,wherein the protein comprises one or more of the following conservedmotifs: CAXDDERG (SEQ ID NO:89), AKLSDFGLAR (SEQ ID NO:90), YELITGR(R/K)(SEQ ID NO:91), and RPKMSEV (SEQ ID NO:92).

5.2 Production of Transgenic Plants with Enhanced Growth and/or Yield

Another aspect of the present invention provides methods of producingplants with enhanced growth and/or yield. In one embodiment, the methodcomprises: transforming a plant or plant cell with a nucleic acidmolecule comprising a plant kinase and/or phosphatase gene of thepresent invention. In one embodiment, the method comprisesoverexpressing said kinase and/or phosphatase gene in the plant or plantcell. In one embodiment, the present invention further comprises:regenerating, from said transformed plant or plant cell, a plant havingenhanced growth and/or yield.

The term “overexpressing,” “overexpression,” or any of the grammaticalvariations thereof (e.g., over-expressing, over-expression) refers to anincrease in the level of expression of a gene, or the level of a proteinproduct encoded by a gene, wherein such increase is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or200%, when compared to cells of the same type in a wild-type plant ofthe same species cultivated under the same conditions.

In one embodiment, the method further comprises: transforming a plant ora plant cell with a nucleic acid molecule comprising an AtPAP2 gene. Incertain embodiments, the method comprises overexpressing the AtPAP2 genein the plant or plant cell. In one embodiment, the AtPAP2 gene comprisesSEQ ID NO: 73. In certain embodiments, the AtPAP2 gene comprises anucleic acid molecule having a nucleic acid molecule having sequencehaving at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%,or 99% identity with SEQ ID NO: 73.

In one embodiment, the method further comprises: transforming a plant ora plant cell with a nucleic acid molecule encoding AtPAP2 phosphatase.In certain embodiment, the method comprises overexpressing the nucleicacid molecule encoding AtPAP2 phosphatase in the plant or plant cell. Inone embodiment, AtPAP2 phosphatase comprises SEQ ID NO: 74. In certainembodiments, AtPAP2 phosphatase comprises an amino acid molecule havingan amino acid nucleic acid sequence having at least 65%, 70%, 75%, 80%,85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 74.

Provided herein are transgenic plants with enhanced growth and/or yield.In certain embodiments, the transgenic plant comprises a nucleic acidmolecule having a nucleic acid sequence selected from SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 93, 95, 97, 99 or 101. In certain embodiments, thenucleic acid molecule is overexpressed in the transgenic plant whencompared to a wild-type plant of the same species cultivated under thesame conditions. In certain embodiments, the transgenic plant comprisesa nucleic acid molecule having a nucleic acid sequence that is at least65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% identitywith SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99 or 101. Incertain embodiments, the nucleic acid molecule is overexpressed in thetransgenic plant when compared to a wild-type plant of the same speciescultivated under the same conditions. In certain embodiments, thetransgenic plant comprises a nucleic acid molecule that is a homologue,derivative, or variant of a nucleic acid molecule derived from thenucleic acid molecule having a nucleic acid sequence selected from SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99 or 101. In certainembodiments, the nucleic acid molecule is overexpressed in thetransgenic plant when compared to a wild-type plant of the same speciescultivated under the same conditions.

In certain embodiments, the transgenic plant comprises a nucleic acidthat encodes a protein having an amino acid sequence selected from SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98, 100 or 102. In certainembodiments, the nucleic acid molecule is overexpressed in thetransgenic plant when compared to a wild-type plant of the same speciescultivated under the same conditions. In certain embodiments, thetransgenic plant comprises a nucleic acid that encodes a protein havingan amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%,93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 94, 96, 98, 100 or 102. In certain embodiments, the nucleic acidmolecule is overexpressed in the transgenic plant when compared to awild-type plant of the same species cultivated under the sameconditions. In certain embodiments, the transgenic plant comprises anucleic acid that encodes a protein that is a homologue, derivative, orvariant of a protein derived from the peptide having an amino acidsequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94, 96, 98,100 or 102. In certain embodiments, the nucleic acid molecule isoverexpressed in the transgenic plant when compared to a wild-type plantof the same species cultivated under the same conditions.

In certain embodiments, the transgenic plant comprises a protein havingan amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,94, 96, 98, 100 or 102. In certain embodiments, the level of the proteinin the transgenic plant is higher than that of a wild-type plant of thesame species cultivated under the same conditions. In certainembodiments, the transgenic plant comprises a protein having an aminoacid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%,96%, 97%, 98% or 99% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,94, 96, 98, 100 or 102. In certain embodiments, the level of the proteinin the transgenic plant is higher than that of a wild-type plant of thesame species cultivated under the same conditions. In certainembodiments, the transgenic plant comprises a protein that is ahomologue, derivative, or variant of a protein derived from the peptidehaving an amino acid and sequence selected from SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 94, 96, 98, 100 or 102. In certain embodiments, the level ofthe protein in the transgenic plant is higher than that of a wild-typeplant of the same species cultivated under the same conditions.

In addition, the present invention provides transgenic plant cellstransformed with a nucleic acid molecule of the present invention. Inone embodiment, the invention provides transgenic plant cells comprisinga kinase or phosphatase nucleic acid molecule of the invention. Incertain embodiments, the nucleic acid molecule is overexpressed in thetransgenic plant cells when compared to plant cells of the same type ina wild-type plant of the same species cultivated under the sameconditions. In another embodiment, the invention provides transgenicplant cells comprising a kinase or phosphatase protein of the invention,wherein the level of said protein in the transgenic plant cells ishigher (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, or 200% higher) than that of plant cells ofthe same type in a wild-type plant of the same species cultivated underthe same conditions.

In certain embodiments, the transgenic plant comprises a nucleic acidmolecule encoding a phosphatase having an amino acid sequence selectedfrom SEQ ID NO: 2, 4, 6, 8, 10, or 94 and/or a kinase selected from SEQID NO: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 96, 98, 100 or 102. In another embodiment, thetransgenic plant comprises a nucleic acid molecule encoding aphosphatase having an amino acid sequence selected from SEQ ID NO: 2, 4,6, 8, 10, or 94 and/or a kinase selected from SEQ ID NO: 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 96, 98,100 or 102. In certain embodiment, all or a portion, particularly anN-terminal portion, of amino acid residues 1 to 80, preferably all or aportion of amino acid residues 1 to 30, are replaced by a heterologousplant signal peptide by genetic engineering. In such a transgenic plant,the phosphatases or kinases are directed to variousorganelles/compartments of the cells.

In certain embodiments, the present invention provides chimeric geneconstructs for genetic modification of plants to increase growth rateand to improve yield. In a specific embodiment, the chimeric geneconstructs comprise a nucleic acid molecule having the nucleic acidsequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97, 99 or 101.In another specific embodiment, the chimeric gene constructs comprise asequence that hybridizes under stringent conditions to a nucleic acidmolecule comprising a nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 93, 95, 97, 99 or 101, or a complement thereof, wherein thenucleic acid sequence encodes a protein or a polypeptide that exhibitsat least one structural and/or functional feature of the polypeptidesand enhances plant growth and/or yield.

The phosphatase or kinase-coding sequence is operatively linked toupstream and downstream regulatory components, preferably heterologousto the phosphatase or kinase sequence, such as for example, CMV 35Spromoter, which acts to cause expression of the gene (production of theenzyme) in plant cells (see FIG. 3). Preferably, when a constructcomprising a gene encoding a phosphatase or kinase of the presentinvention is introduced into plant cells by a conventionaltransformation method, such as microparticle bombardment, Agrobacteriuminfection, or microinjection, the gene is expressed in the cells underthe control of the regulatory sequences. The expressed phosphataseinteracts with the biosynthetic machinery that is naturally present inthe plant cells to alter the carbon metabolism. By altering the carbonmetabolism, the method of the present invention promotes the growth rateof the plant, resulting in faster growth rate and higher yield. As aresult, the time required for the maturation of the plant and the timerequired for flowering is shortened. Also provided are methods forincreasing growth rate and yield of plants, comprising the step ofinserting into such plant cells, or cells of such whole plants, achimeric gene construct.

In one specific embodiment, Arabidopsis is genetically modified byintroducing an overexpression construct comprising nucleic acidmolecules encoding a growth-promoting phosphatase or kinase of thepresent invention.

In an embodiment, the growth-promoting phosphatase and kinase genes arederived from Arabidopsis. As shown in the examples, transgenicArabidopsis plants with over-expression of NG6, NG21, NG24, NG28, and/orNG32 have enhanced growth and/or yield, when compared to wild-typeArabidopsis plants (see Table 5, and FIGS. 5 and 6).

In one embodiment, a transgenic plant overexpressing a nucleic acidcomprises the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 93, 95, 97,99 or 101 or homologues thereof, wherein the nucleic acid moleculeencodes polypeptides or proteins of the invention.

5.3 Homologues, Derivatives, and Variants of Kinases and Phosphatases

The present invention also provides homologues, derivatives, andvariants of kinases and phosphatases of the present invention; nucleicacid molecules encoding the polypeptides and homologues, derivatives,and variants; vectors, plant cells and transgenic plants comprisingthese nucleic acid molecules; and uses thereof for promoting plantgrowth and/or yield. The homologues, derivatives and variants of kinasesand phosphatases are derived from the wild-type kinases andphosphatases, respectively. The methods of deriving the homologues,derivatives and variants are well known in the art which include routineconventional techniques of chemical modifications of amino acid residuesor using molecular biology and recombinant DNA manipulation andproduction. Such techniques are available to the skilled artisan inlaboratory manuals such as Sambrook and Russell, Molecular cloning: ALaboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (2001).

In one embodiment, a homologue of the nucleic acid or polypeptidemolecule of the present invention includes: (i) a polypeptide with atleast about 65%, at least about 70%, at least about 80%, at least about90%, or at least about 98% sequence identity of the polypeptide of theinvention; (ii) a polypeptide encoded by a nucleotide sequence that isat least about 65%, at least about 70%, at least about 80%, at leastabout 90%, or at least about 98% identical to one or more of thenucleotide sequences encoding a polypeptide of the invention, or afragment thereof; (iii) a polypeptide encoded by a nucleotide sequencethat hybridizes, under stringent conditions, to a nucleotide sequence ofthe present invention; (iv) a polypeptide having an amino acid sequencethat is at least about 65%, at least about 70%, at least about 80%, atleast about 90%, or at least about 98% identical to a polypeptide of thepresent invention, and wherein the polypeptide of the invention isconservatively substituted; (v) a nucleic acid sequence encoding anamino acid sequence that is at least about 70%, at least about 80%, atleast about 90%, or at least about 98% identical to a polypeptide of thepresent invention and wherein the polypeptide of the invention isconservatively substituted; and (vi) a fragment of a polypeptidedescribed in (i) through (iv), wherein the polypeptide fragment has atleast 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550,600, 650, 700, or 750 contiguous amino acid residues of a polypeptide ofthe invention.

In one embodiment, a homologue polypeptide has an amino acid sequencethat is at least about 65%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or at least about 98% identical toa kinase or phosphatase of the present invention. In one embodiment, thehomologue polypeptide is obtained by conservative substitution.

In one aspect, the homologues derivatives and variants are derived fromthe wild type kinase and phosphatase by substitution, deletion,insertion of one or more nucleic acid in a nucleic acid molecule or oneor more amino acid residues in an amino acid molecule. The term“derived” as used herein includes the modifications of a wild typenucleic acid molecule or amino acid molecule as described below. Forexample, non-natural amino acids can be substituted for the amino acidsof the kinases and phosphatases so long as the kinases and phosphataseshaving the substituted amino acids retain substantially the samefunctional activity as the kinases and phosphatases in which amino acidshave not been substituted. Those having skill in the art will recognizethat mutations can be made to polynucleotides encoding protein andpeptides, or complementary thereto, and that such mutations do not causestructural changes that affect functionality.

Conservative substitutions whereby a modified protein or polypeptide ofthe present invention having an amino acid of one class is replaced withanother amino acid of the same class fall within the scope of thesubject invention so long as the modified protein or polypeptide havingthe substitution still retains substantially the same functionalactivity as the protein or polypeptide that does not have thesubstitution. For instance, amino acid residue of any of the following11 groups may be conservatively substituted with another amino acid ofthe same group: (1) acidic (negatively charged) amino acids, such asaspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids, such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine; (5) amino acids havingaliphatic side chains, such as glycine, alanine, valine, leucine, andisoleucine; (6) amino acids having aliphatic-hydroxyl side chains, suchas serine and threonine; (7) amino acids having amide-containing sidechains, such as asparagine and glutamine; (8) amino acids havingaromatic side chains, such as phenylalanine, tyrosine, and tryptophan;(9) amino acids having basic side chains, such as lysine, arginine, andhistidine; (10) amino acids having sulfur-containing side chains, suchas cysteine and methionine; and (11) amino acids having similar geometryand hydrogen bonding patterns, such as aspartic acid, asparagine,glutamic acid and glutamine.

Examples of non-natural amino acids include, but are not limited to,ornithine, citrulline, hydroxyproline, homoserine, phenylglycine,taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-aminohexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline,cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, C-methyl amino acids, N-methyl aminoacids, and amino acid analogues in general. Non-natural amino acids alsoinclude amino acids having derivatized side groups. Furthermore, any ofthe amino acids in the protein can be of the D (dextrorotary) form or L(levorotary) form.

The structure of a polypeptide can be determined by methods known tothose skilled in the art, including but not limited to, X-raycrystallography, nuclear magnetic resonance, and crystallographicelectron microscopy. A sequence having sequence homology can be madeusing standard molecular biology techniques, including site-directedmutagenesis and by insertion or deletion of sequences.

In one aspect, the homologues, derivatives and variants are derived fromthe wild type kinase and phosphatase. In certain embodiments, providedherein are derivatives of the disclosed polypeptides. For example, butnot by way of limitation, derivatives may include peptides or proteinsthat have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques including, but not limited to,specific chemical cleavage, acetylation, formylation, etc. Additionally,the derivative may contain one or more non-classical amino acids. Thesubject invention also concerns variants of the polynucleotides of thepresent invention. Variant sequences include those sequences wherein oneor more nucleotides of the sequence have been substituted, deleted,and/or inserted.

The nucleotides that can be substituted for natural nucleotides of DNAhave a base moiety that can include, but is not limited to, inosine,5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine,5-methylcytosine, and tritylated bases. The sugar moiety of thenucleotide in a sequence can also be modified and includes, but is notlimited to, arabinose, xylulose, and hexose. In addition, the adenine,cytosine, guanine, thymine, and uracil bases of the nucleotides can bemodified with acetyl, methyl, and/or thio groups. Sequences containingnucleotide substitutions, deletions, and/or insertions can be preparedand tested using standard techniques known in the art.

Unless otherwise specified, as used herein percent sequence identityand/or similarity of two sequences can be determined using the algorithmof Karlin and Altschul (1990), modified as in Karlin and Altschul(1993). Such an algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul et al. (1990). BLAST searches can be performed withthe NBLAST program, score=100, wordlength=12, to obtain sequences withthe desired percent sequence identity. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be used as described in Altschulet al. (1997). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (NBLAST and XBLAST) can beused. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide moleculeshaving sequences which are sufficiently homologous with thepolynucleotide sequences exemplified herein so as to permithybridization with that sequence under standard stringent conditions andstandard methods (Maniatis et al., 1982).

In one embodiment, the present invention further provides isolatednucleic acid molecules that comprise, or consist of, at least about 550,at least about 600, at least about 650, at least about 700, at leastabout 750, at least about 800, at least about 850, at least about 900,at least about 950, at least about 1000, at least about 1050, at leastabout 1100, at least about 1150, at least about 1200, at least about1250, at least about 1300, or at least about 1350 contiguous nucleotidesof a nucleic acid molecule of the present invention.

In another embodiment, an isolated nucleic acid molecule encodes avariant of a polypeptide whose amino acid sequence has been modified bygenetic engineering so that biological activities of the polypeptidesare either enhanced or reduced, or the local structures thereof arechanged without significantly altering the biological activities. Aminoacid modifications can be made by methods known in the art.

In one embodiment, the present invention embodies isolated nucleic acidmolecules that hybridize, under stringent conditions, to nucleic acidmolecules having the nucleic acid sequence comprising SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 93, 95, 97, 99 or 101, or homologues thereof. In certainembodiments, the nucleic acid molecules encode proteins or polypeptidesthat exhibit at least one structural and/or functional feature of thepolypeptides of the invention (e.g. enhance plant growth and/or yield).

A further embodiment includes methods for preparing a polypeptide asprovided herein by recombinant DNA technology. In one embodiment, thepreparation method comprises culturing host cells containing arecombinant expression vector encoding a polypeptide as provided herein,or a nucleotide sequence encoding a polypeptide as provided hereinoperably linked to a heterologous promoter, and producing thepolypeptide as provided herein.

5.4 Vectors and Expression Constructs

Another embodiment includes nucleic acid molecules suitable for use asprimers or hybridization probes for the detection of nucleic acidsencoding a phosphatase or kinase polypeptide as provided herein or othersequences.

Yet another embodiment includes vectors, e.g., recombinant expressionvectors, comprising a nucleic acid molecule as provided herein.Furthermore, host cells containing such a vector or engineered tocontain and/or express a nucleic acid molecule as provided herein andhost cells containing a nucleotide sequence as provided herein operablylinked to a heterologous promoter are disclosed.

As used herein, the term “expression construct” refers to a combinationof nucleic acid sequences that provides for transcription of an operablylinked nucleic acid sequence. In general, operably linked components arein contiguous relation.

Expression constructs of the invention will also generally includeregulatory elements that are functional in the intended host cell inwhich the expression construct is to be expressed. Regulatory elementsinclude promoters, transcription termination sequences, translationtermination sequences, enhancers, and polyadenylation elements.

An expression construct as provided herein can comprise a promotersequence operably linked to a polynucleotide sequence encoding apeptide. Promoters can be incorporated into a polynucleotide usingstandard techniques known in the art. Multiple copies of promoters ormultiple promoters can be used in an expression construct. In apreferred embodiment, a promoter can be positioned about the samedistance from the transcription start site as it is from thetranscription start site in its natural genetic environment. Somevariation in this distance is permitted without substantial decrease inpromoter activity. A transcription start site is typically included inthe expression construct.

Unique restriction enzyme sites can be included at the 5′ and 3′ ends ofthe expression construct to allow for insertion into a polynucleotidevector. As used herein, the term “vector” refers to any genetic element,including for example, plasmids, cosmids, chromosomes, phage, virus, andthe like, which is capable of replication when associated with propercontrol elements and which can transfer polynucleotide sequences betweencells. Vectors contain a nucleotide sequence that permits the vector toreplicate in a selected host cell.

The term “operably linked,” as used herein, refers to when transcriptionunder the control of the “operably linked” promoter produces afunctional messenger RNA, translation of which results in the productionof the polypeptide encoded by the DNA operably linked to the promoter.

5.5 Fusion Proteins

Also provided herein are fusion proteins. In one embodiment, thepolypeptides as provided herein, or fragments thereof, are recombinantlyfused or chemically conjugated (e.g., covalent and non-covalentconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion can be direct, or may occur throughlinker sequences.

In one embodiment, the fusion protein comprises a polypeptide fused to aheterologous signal sequence at its N-terminus. For example, the signalsequence naturally found in the polypeptide can be replaced by a signalsequence that is derived from a heterologous origin. Various signalsequences are commercially available.

In another embodiment, a polypeptide can be fused to tag sequences,e.g., a hexa-histidine peptide, among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA, 86:821-824, for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other examples of peptidetag include the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell, 37:767), and the “flag” tag (Knappik et al., 1994, Biotechniques,17(4):754-761). These tags are useful for purification of recombinantlyproduced polypeptides.

Fusion proteins can be produced by standard recombinant DNA techniquesor by protein synthetic techniques, e.g., by use of a DNA synthesizer.For example, a nucleic acid molecule encoding a fusion protein can besynthesized by conventional techniques including, for example, automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers, which give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., JohnWiley & Sons, 1992).

The nucleotide sequence encoding a fusion protein can be inserted intoan appropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. In a specific embodiment, the expression of afusion protein is regulated by an inducible promoter.

In another embodiment, the present invention provides methods fordetecting the presence, activity or expression of a polypeptide of theinvention or similar polypeptide in a biological material, such ascells, or culture media. The increased or decreased activity orexpression of the polypeptide in a sample relative to a control samplecan be determined by contacting the biological material with an agentthat can detect directly or indirectly the presence, activity orexpression of the polypeptide. In a particular embodiment, such an agentis an antibody or a fragment thereof which immunospecifically binds toone of the disclosed polypeptides.

In a still another embodiment, a fusion protein comprising a bioactivemolecule and one or more domains of a disclosed polypeptide or fragmentthereof is provided. In particular, fusion proteins comprising abioactive molecule recombinantly fused or chemically conjugated(including both covalent and non-covalent conjugations) to one or moredomains of a disclosed polypeptide or fragments thereof.

5.6 Preparation of Transgenic Plants

Genetic engineering of plants can be achieved in several ways. The mostcommon method is Agrobacterium-mediated transformation. In this method,A. tumefaciens, which naturally infects plants by insertingtumor-causing genes into a plant's genome, is genetically altered.Selected genes can be engineered into the T-DNA of the bacterial Ti(tumor-inducing) plasmid of A. tumefaciens in laboratory conditions sothat they become integrated into the plant chromosomes when the T-DNA istransferred to the plant by the bacteria's own internal transfermechanisms.

The only essential parts of the T-DNA are its two small (25 base pair)border repeats, at least one of which is needed for planttransformation. The bacterial genes encoding for plant hormones thatpromote tumor growth are excised from the T-DNA and replaced with asequence of DNA that typically contains: a selectable marker (e.g. anantibiotic-resistance gene; usually kanamycin resistance), a restrictionsite—a site with a specific sequence of nucleotides where a restrictionenzyme will cut the DNA, and the desired genes to be incorporated intothe plant (B. Tinland, 1996. The integration of T-DNA into plantgenomes. Trends in Plant Science 1,178-184; D. Grierson (ed.) 1991.Plant Genetic Engineering. Blackie, Glasgow).

Agrobacterium can be added to plant protoplasts (plant cells with cellwalls removed) in culture; the plant protoplasts then regenerate cellwalls at which point non-transformed plants are killed with antibioticsfor which the transformed plants have been given resistance genes.Plantlets are then regenerated from the surviving transformed cellsusing standard plant tissue culture techniques.

In an alternative technique, sterile disks or fragments of vegetativeportions of plants are placed in liquid culture medium withAgrobacterium, and then hormones are used to induce rooting, therebyregenerating plantlets grown on selection media. Another technique fordelivering genes is possible for some plants such as Arabidopsis, wherethe Agrobacterium or even “naked” DNA can be infused through the seedcoat to cause transformation (Clough S J and Bent A F, 1998. Floral dip:a simplified method for Agrobacterium-mediated transformation ofArabidopsis thaliana. Plant J 16:735-43).

The biolistic method for genetic engineering of plants was developedmore recently and is becoming more widely employed. In this method, verysmall particles (microprojectiles) of tungsten or gold coated withbiologically active DNA are propelled at high-velocities into plantcells using an electrostatic pulse, air pressure, or gunpowderpercussion. As the particles pass through the cell, the DNA dissolvesand can then integrate into the genome of that cell and its progeny.This method can produce stable transformants (Christou, P., et al.,1988. Stable transformation of soybean callus by DNA-coated goldparticles, Plant Physiology 87:671-674). The method can be practiced onwhole plants and is particularly effective on meristematic tissue. It isalso capable of delivering DNA either to the nucleus or intomitochondria (Johnston, S. A., et al., 1988. Mitochondrialtransformation in yeast by bombardment with microprojectiles (Science240,1538-41) and chloroplasts (Svab, Z., et al., 1990, Stabletransformation of plastids in higher plants, Proc Natl Acad Sci. USA 87,8526-8530).

The electroporation method of plant genetic engineering has met withless success. In this technique, protoplasts in culture take up pure DNAwhen treated with certain membrane-active agents or withelectroporation—a rapid pulse of high-voltage direct current. Once theDNA enters the protoplast, it can be integrated into the cells genome.Standard tissue culture techniques are then used to regeneratetransgenic plants.

The microinjection method of plant genetic engineering is perhaps themost difficult. In this method, DNA is microinjected into target plantcells using very thin glass needles in a method similar to that usedwith animals. The technique is laborious, ineffective, and impracticalfor generating large numbers of transgenic plants.

It is within the ability of a skilled artisan to select known methodsfor producing genetically engineering plants, taking into accountvarious factors such as the targeted plant species and which methodshave been proven effective therein.

5.7 Preparation of Antibodies

In one aspect, provided herein are antibodies against the kinase andphosphatase. Antibodies which specifically recognize one of thedescribed phosphatase polypeptides or fragments thereof can be used fordetecting, screening, and isolating the polypeptide that is providedherein or fragments thereof, or similar sequences that encode similarenzymes from other organisms. For example, an antibody whichimmunospecifically binds a protein or protein fragments thereof can beused for various in vitro detection assays, including enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays, Western blot, etc., forthe detection of the polypeptide that is provided herein or fragments,derivatives, homologues, or variants thereof, or similar moleculeshaving the similar enzymatic activities as the phosphatase and/or kinasepolypeptides.

Embodiments further provide antibodies that immunospecifically bind apolypeptide that is provided herein. Such antibodies include, but arenot limited to, antibodies from various animals, humanized, chimeric,polyclonal, monoclonal, bi-specific, multi-specific, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs,fragments containing a VL or VH domain or a complementary determiningregion (CDR), wherein the antibody or antibody fragmentimmunospecifically binds to a polypeptide that is provided herein.

Antibodies specific for the described phosphatase polypeptides can begenerated by any suitable method known in the art. Once an antibodymolecule has been produced, it may then be purified by any method knownin the art for purification of an immunoglobulin molecule, for example,by chromatography (e.g., ion exchange, affinity, particularly byaffinity for the specific antigen after Protein A or Protein Gpurification, and sizing column chromatography), centrifugation,differential solubility, or by any other standard techniques for thepurification of proteins. Further, the antibodies or fragments thereofmay be fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

Antibodies fused or conjugated to heterologous polypeptides may be usedin in vitro immunoassays and in purification methods (e.g., affinitychromatography) well known in the art. See e.g., PCT publication NumberWO 93/21232; EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99;U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; andFell et al., 1991, J. Immunol. 146:2446-2452, which are incorporatedherein by reference in their entireties.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the describedpolypeptides or fragments, derivatives, homologues, or variants thereof,or similar molecules having the similar enzymatic activities as thepolypeptide of the invention. Such solid supports include, but are notlimited to, glass, cellulose, polyacrylamide, nylon, and polystyrene.

5.8 Detection Assays

An exemplary method for detecting the presence or absence of anover-expressed phosphatase/kinase polypeptide or an insertedphosphatase/kinase-encoding nucleic acid in a biological sample involvesobtaining a biological sample from various sources and contacting thesample with a compound or an agent capable of detecting a polypeptide ornucleic acid (e.g., mRNA, genomic DNA) such that the presence of aheterologous polypeptide or nucleic acid is detected in the sample.

An exemplary agent for detecting mRNA or genomic DNA encoding aninserted phosphatase polypeptide is a labeled nucleic acid probe capableof hybridizing to mRNA or genomic DNA encoding any of the describedphosphatase and kinase polypeptides. The nucleic acid probe can be, forexample, a full-length cDNA, such as the nucleic acid of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 93, 95, 97, 99, 101 or a portion thereof, such as anoligonucleotide of at least one of at least about 15, at least about 20,at least about 25, at least about 30, at least about 50, at least about100, at least about 250, at least about 500, or more nucleotides inlength and sufficient to specifically hybridize under stringentconditions to a mRNA or genomic DNA encoding a polypeptide of theinvention.

An exemplary agent for detecting an over-expressed phosphatase/kinasepolypeptide is an antibody capable of binding to a phosphatase/kinasepolypeptide product of an inserted gene, preferably an antibody with adetectable label. Antibodies can be polyclonal and monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used.

The term “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

The detection method can be used to detect mRNA, protein, or genomic DNAin a sample in vitro as well as in vivo. For example, in vitrotechniques for detection of mRNA include Northern hybridizations and insitu hybridizations. In vitro techniques for detection of a heterologouspolypeptide include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a heterologouspolypeptide include introducing into a subject organism a labeledantibody directed against the polypeptide. For example, the antibody canbe labeled with a radioactive marker whose presence and location in thesubject organism can be detected by standard imaging techniques,including autoradiography.

In a specific embodiment, the methods further involve: 1) obtaining acontrol sample from a control subject, 2) contacting the control samplewith a compound or agent capable of detecting an over-expressedpolypeptide product, or the mRNA transcription product, or genomic DNAencoding an inserted phospatase gene, such that the presence of thepolypeptide or mRNA or genomic DNA encoding the phosphatase polypeptideis detected in the sample, and 3) comparing the level of thephosphatase/kinase polypeptide or mRNA or genomic DNA encoding thepolypeptide in a control sample with the level of the polypeptide ormRNA or genomic DNA encoding endogenous phosphatase polypeptides in thetest sample.

5.9 Applications of Transgenic Plants

The transgenic plants generated can have many useful applications,including in food, feed, biomass, biofuels (starch, cellulose, seedlipids) and wood pulp industry. The enhanced growth rate of thetransgenic plants can provide additional carbon dioxide fixation perhectare of land per year, and, thus is useful for generating carboncredits.

6.0 EXAMPLES

Following are examples that illustrate embodiments for practicing theinvention. These examples should not be construed as limiting. Unlessotherwise noted, all percentages are by weight, all solvent mixtureproportions are by volume, all temperatures are in Centigrade, and allpressure is at or near atmospheric pressure.

6.1 Screening of Grow-Promoting NG Genes

Two independent AtPAP2 overexpression lines (OE7 and OE21, homozygous T3plants), an AtPAP2 T-DNA mutant line that cannot express the full lengthAtPAP2, and the wild-type Arabidopsis (Col-0) were employed formicroarray analysis. The AtPAP2 overexpression lines (OE7 and OE21,homozygous T3 plants), the AtPAP2 T-DNA mutant line, and the wild-typeArabidopsis (Col-0) line have been disclosed by the present inventor inU.S. patent application Ser. No. 12/640,674 (U.S. Patent ApplicationPublication No. 2010/0159065), which is hereby incorporated by referencein its entirety.

Briefly, seeds were germinated on MS medium supplemented with 2% (w/v)sucrose, grew in a growth room under 12 hour-light/12 hour-dark cycle at22° C. for 10 days, and were then transferred to soil and grew in agrowth chamber under a 16-hour light (22° C.) and 8-hour dark (18° C.)cycle. Shoots of 20-day-old Arabidopsis (WT, T-DNA, OE7 and OE21) priorto bolting were collected in the middle of day (4 plants/line/tube, 3biological triplicates/line, 3 tubes/line) and ground in liquidnitrogen. RNA extraction was performed with on-column DNase digestionaccording to the manufacturer's instruction (RNeasy Plant Mini Kit, Cat.No. 74904, Qiagen). Total RNA was dissolved in DEPC water and quantifiedby the Bioanalyzer 2100 (Agilent Technologies, Boblingen, Germany).Double strand DNA synthesis and Cy 3 labeling from three biologicalreplicates were performed by NimbleGen Systems, Inc. (Madison, Wis.).Statistical analyses of normalized microarray data (RMA algorithm,quantile normalization) and drawing of scatter plots, heatmaps wereperformed using ArrayStar 3.0 (DNASTAR, Madison, Wis.). Identificationof GO and classification were carried out using software available fromTAIR database and KEGG pathway database. In all three replications,genes were considered to be significantly regulated if their fold changevalues were positively or negatively beyond 1.3 (p<0.05).

20-day-old plants did not show any differences in appearance so that anydifferences in gene expression between the lines were not due todifference in developmental stage or additional tissues (e.g.inflorescence). The transcripts levels of 30360 genes in shoots weredetermined using the Arabidopsis Genome NimbleGen chips. The averagehybridization signals detected in each line were normalized from thelog2 average signal and compared with the signal strengths in thewild-type Arabidopsis.

An overview of the expression data of OE7, OE21 and T-DNA plants versuswild-type control is presented as a heat map (FIG. 1) and scatter plots(FIG. 2) that show a linear bias in the graphs. Gene expression patternsin transgenic shoots are different comparative to their wild-typecontrols.

The data show that AtPAP2 overexpression altered expression levels ofother genes, nearly half of which have not been characterized yet.AtPAP2 overexpression lines exhibit more dramatic changes in geneexpression than the AtPAP2 T-DNA line.

Differentially expressed genes are identified using P-value<0.05 andfold change>1.3 as the cutoff, and the results show that the expressionof about 6312, 7831, and 672 genes in the shoots of OE7, OE21 and T-DNAlines are significantly altered. An overall view of the altered genes inthe heat map (FIG. 1) revealed that most genes were down-regulated inthe fold change>=2.0. In addition, the fold change in expression levelsis smaller in up-regulated genes than in down-regulated genes.

Based on the microarray data, 33 putative phosphatase and kinase geneswere selected, and were introduced into Arabidopsis to produceoverexpression lines. The results show that the overexpression of NG6,NG21, NG24, NG28 and NG32 in Arabidopsis promotes the growth ofArabidopsis and increases seed yield (see Example 2). The expressionlevel of the five growth-promoting NG genes in the AtPAP2 OE lines andT-DNA lines are shown in Table 1.

TABLE 1 Microarray data of the 5 growth-promoting genes in AtPAP2overexpression lines (OE7, OE21), T-DNA line and wild type (WT)Arabidopsis. WT- T-DNA OE7 OE21 OE7/WT OE21/WT T-DNA/WT Gene NG No. AGIcode Mean Mean Mean Mean Fold Fold Fold Description NG 6 AT1G05000 637533 976 1131 1.52* 1.78** 0.83 Protein phosphatase NG21 AT1G13350 24062151 3543 3441 1.47* 1.43* 0.89 Protein kinase NG24 AT1G28390 778 7101853 1915 2.37** 2.42** 0.91 Protein kinase NG28 AT3G24660 2514 18393313 4422 1.32** 1.74** 0.73 Protein kinase NG32 AT5G03320 1325 10631884 2053 1.43* 1.56** 0.80 Protein kinase

6.2 Production of NG Overexpression Lines in Arabidopsis

To create transgenic NG gene overexpressing lines, the full lengthcoding region of each NG gene's cDNA was amplified by PCR using thefollowing primers (Table 2). The PCR products were inserted into thepCXSN vector with classical TA cloning method (FIG. 3).

TABLE 2 Primers used for to amplify the full CDS of the aimed NG genesGene name Sequence(5′-3′) NG6 Forward Primer5′-TCGAGCTAGCATGAAGCTTGTGGAGAAGAC-3′ (SEQ ID NO: 51) Reverse Primer5′-CGACGAGCTCTTACCTGATGGAACAAGAG-3  (SEQ ID NO: 52) NG21 Forward Primer5′-ATGGTGAGTGACAAGCATGTAG-3′ (SEQ ID NO: 53) Reverse Primer5′-TCACTTGCCCGTGATGAATG-3′ (SEQ ID NO: 54) NG24 Forward Primer5′-ATGGGTTATCTCTCTTGCAAC-3′ (SEQ ID NO: 55) Reverse Primer5′-TCAGTATCTCTTCCGCGACG-3′ (SEQ ID NO: 56) NG28 Forward Primer5′-ATGGGCATGGAAGCTTTGAG-3′ (SEQ ID NO: 57) Reverse Primer5′-TCAAAATGGAGTTTCGGCGT-3′ (SEQ ID NO: 58) NG32 Forward Primer5′-ATGAAATGCTTCTTATTCCC-3′ (SEQ ID NO: 59) Reverse Primer5′-TCAACAAGCTCTCACATTCT-3′ (SEQ ID NO: 60)

The vector was introduced into Agrobacterium tumefaciens strain GV3101and then transformed by the floral dip method (Clough and Bent, 1998)into wild-type Col-0 to generate NG-overexpressing lines. Through twogenerations of selection on MS agar plate with 30 mg/1 hygromycin,homologous NG transgenic lines were obtained. The resistant plants weretransferred to soil to grow to maturity, and their transgenic status wasconfirmed by qRT-PCR analysis.

6.3 Confirmation of Overexpression of NG Genes in Transgenic Plants

The transcription levels of the NG genes in the hygromycin resistant,homologous T3 overexpression lines were confirmed by quantitative RealTime-PCR. Total RNA was extracted from 10-day-old seedlings grown onMurashige and Skoog (MS) with 3% (w/v) sucrose using the TRIzol RNAisolation method with DNase I treatment. cDNAs were generated usingSuperscript III reverse transcriptase (Invitrogen, Carlsbad, Calif.,USA) using an oligol 5 dT primer. Two gene-specific primers were used toamplify the 80-150 bp coding region of each NG gene. The ACTIN primerswere used for control experiment. As shown in FIG. 4, the transcriptlevels of each overexpression line were consistently higher than theirrespective expression levels in the wild-type.

TABLE 3 Primers used in the quantitative RT-PCR NG6 Forward Primer5′-TGTGCCCGGAGCCCTACC-3′ (SEQ ID NO: 61) Reverse Primer5′-CTTTCAGTGCCATGCGGATTTT-3 (SEQ ID NO: 62) NG21 Forward Primer5′-GGCACAAGTCCCGTCATCACC-3′ (SEQ ID NO: 63) Reverse Primer5′-TCCCCAATCCCTTCTTTTCCTA-3′ (SEQ ID NO: 64) NG24 Forward Primer5′-GCCGCCGTCAAGAGAACAAC-3′ (SEQ ID NO: 65) Reverse Primer5′-CTCCGGTGGTCAACGCAGTAA-3′ (SEQ ID NO: 66) NG28 Forward Primer5′-TGTTGTTGTGGCCTCGTTGTTA-3′ (SEQ ID NO: 67) Reverse Primer5′-CTTTCCTTCACCGCCTTCTTTC-3′ (SEQ ID NO: 68) NG32 Forward Primer5′-AAGCTTTCGGATTTCGGTTTG-3′ (SEQ ID NO: 69) Reverse Primer5′-TGGCCTTCTTCCTGTAATGAGC-3′ (SEQ ID NO: 70) ACTIN Forward Primer5′-CCCGCTATGTATGTCGC-3′ (SEQ ID NO: 71) Reverse Primer5′-AAGGTCAAGACGGAGGAT-3′ (SEQ ID NO: 72)

6.4 Growth Phenotypes of NG Gene Over-Expression Lines

Arabidopsis seeds were soaked in water at 4° C. for 3 days. The seedswere surface sterilized and sown on MS medium supplemented with 3% (w/v)sucrose for 10 days. Seedlings with 2 rosette leaves of the same sizewere transferred to soil under Long Day condition (16 h light at 22°C./8 h dark at 18° C.) in a plant growth chamber. Bolting time wasmeasured when the primary inflorescence reached 1 cm above the rosetteleaves. (Liu et al., 2008; Wu et al., 2008).

The inflorescences of NG gene OE lines emerged earlier (4-5 days) thanthe WT at Long Day conditions (Table 4, FIG. 5 and FIG. 6). Thisphenotype observation was repeated at least 3 times and the results oftwo of the experiments are shown here.

TABLE 4 WT NG6 NG21 NG24 NG28 NG32 A. Earlier bolting time of NG OElines (Trial 1) Average bolting 24.4 21.2 20.1 21.4 20.8 19.8 time (Day)SD 1.4 1.0 1.4 0.9 1.0 1.3 N 12 12 12 9 9 9 B. Earlier bolting time ofNG OE lines (Trial 2) Average bolting 24.3 19.2 19 19 18.3 19 time (Day)SD 0.8 0.8 1.1 1 1.0 0.9 N 12 6 6 9 6 6

At maturity (Long Day), the number of inflorescence and the total weightof seeds harvested from each line were recorded. The results of twoseparate experimental trials are shown in Tables 5A and B. The resultsshow that the overexpression of each of the five NG genes (NG6, NG21,NG24, NG28, and NG32) resulted in increased number of inflorescences andseed yield. Compared to that of the wild-type, the seed yield of each NGover-expression line increased 30-50% (Table 5).

TABLE 5A OE lines produced more seeds (Trial 1). Lines Weight of seeds(mg)/plant SD WT(Col-0) 80.4 4.9 NG6 113.6 12.2 NG21 127.8 26.9 NG2499.6 17.3 NG28 130.6 26.7 NG32 135.9 23.5The plants were grown in small black trays (N=6-9).

TABLE 5B OE lines produced more seeds (Trial 2). Lines Weight of seeds(mg)/plant SD WT(Col-0) 142.0 14.6 NG6 190.3 15.7 NG21 180.4 26.3 NG24203.8 20.0 NG28 186.0 39.5 NG32 241.8 23.8The plants were grown in large white cups (N=6-9).

The results show that, when compared to the wild-type, Arabidopsisplants transformed with NG6, NG21, NG24, NG28 and/or NG32 have thefollowing advantageous phenotypes: (1) faster growth rate; (2) higherseed yield.

6.5 Sequence Alignment and Phylogenetic Analysis

All the CDS of 5 NG genes in the Arabidopsis Col-0 ecotype were obtainedfrom the TAIR website. Sequence alignment of each NG gene was retrievedby tblastn program from Plant GDB database and NCBI database using theamino acid sequence of each Arabidopsis NG gene as the bait sequences.Partial sequences recovered were aligned and compared to produce a fulllength coding sequence if feasible. Sequence alignment and phylogenetictree were conducted using MEGA4 (Kumar et al., 2004) and ClustalWprogram. Amino acid sequence comparisons were performed using CLCSequence Viewer 5.1.1.

Those skilled in the art will recognize, or be able to ascertain manyequivalents to the specific embodiments of the invention describedherein using no more than routine experimentation. Such equivalents areintended to be encompassed by the following claims.

All publication, patents and patent applications mentioned in thisspecification are incorporated herein by reference in their entiretiesinto the specification to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

REFERENCES

-   Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method    for Agrobacterium-mediated transformation of Arabidopsis thaliana.    Plant J, 16, 735-743.-   Klabunde, T., Strater, N., Frohlich, R., Witzel, H. and    Krebs, B. (1996) Mechanism of Fe(III)-Zn(II) purple acid phosphatase    based on crystal structures. J. mol. biol., 259, 737-748.-   Klabunde, T. and Krebs, B. (1997) The dimetal center in purple acid    phosphatases. Metal Sites in Proteins and Models, 89, 177-198.-   Li, D., Zhu, H., Liu, K., Liu, X., Leggewie, G., Udvardi, M. and    Wang, D. (2002) Purple acid phosphatases of Arabidopsis thaliana.    Comparative analysis and differential regulation by phosphate    deprivation. J. Biol. Chem., 277, 27772-27781.-   Schenk, G., Ge, Y., Carrington, L. E., Wynne, C. J., Searle, I. R.,    Carroll, B. J., Hamilton, S. and de-Jersey, J. (1999) Binuclear    metal centers in plant purple acid phosphatases: Fe—Mn in sweet    potato and Fe-Zn in soybean. Arch. Biochem Biophys, 370, 183-189.-   United States Patent Application Publication No. 2010/0159065

1. A method to make a transgenic plant having increased rate of plantgrowth and/or elevated plant yields comprising: a) introducing a geneexpression construct into a plant or plant cell, wherein the constructcomprises a nucleic acid molecule encoding a phosphatase or kinase andthe nucleic acid molecule comprises: i) a sequence having at least 85%identity with SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 45, 47, 49, 93, 95, 97, 99, or 101; orii) a sequence encoding a polypeptide having at least 85% identity withSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 46, 48, 50, 94, 96, 98, 100, or 102;  wherein thesequence is operatively linked to upstream and downstream regulatorycomponents comprising a heterologous promoter; and b) overexpressing thenucleic acid molecule in the plant or plant cell, wherein the promoterdrives the overexpression of the nucleic acid molecule.
 2. The method ofclaim 1, wherein the nucleic acid molecule comprises: i) a sequenceselected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 45, 47, 49, 93, 95, 97, 99, or 101; orii) a sequence encoding a polypeptide selected from SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,46, 48, 50, 94, 96, 98, 100, or
 102. 3. The method of claim 1, furthercomprising regenerating, from said transformed plant or plant cell, aplant having enhanced growth and/or yield.
 4. The method of claim 1,wherein plant growth rate is increased.
 5. The method of claim 1,wherein plant yield is increased.
 6. The method of claim 1, wherein theplant is of a genus selected from the group consisting of: Asparagus,Bromus, Hemerocallis, Hordeum, Lolium, Panicum, Pennisetum, Saccharum,Sorghum, Trigonella, Triticum, Zea, Antirrhinum, Arabidopsis, Arachis,Atropa, Brassica, Browallia, Capsicum, Carthamus, Cichorium, Citrus,Chrysanthemum, Cucumis, Datura, Daucus, Digitalis, Fragaria, Geranium,Glycine, Helianthus, Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Majorana,Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis,Pelargonium, Petunia, Ranunculus, Raphanus, Salpiglossis, Senecio,Sinapis, Solanum, Trifolium, Vigna, and Vitis.
 7. The method of claim 1,wherein the plant is of a species of the genus Brassica.
 8. The methodof claim 1, wherein the plant is of a species selected from the groupconsisting of: Glycine max, Zea mays, Solanum tuberosum, Panicumvirgatum, Medicago sativa, and Brassica napus.
 9. The method of claim 1,wherein the plant cell is a seed, stem, shoot, or root cell. 10.(canceled)
 11. The method of claim 1, wherein plant weight, total weightof leaf or seed, total number of inflorescence, carbon metabolism, levelof carbohydrate, amino acid, lipid production or seed yield per plantare increased; or bolting time is earlier, when compared to a wild-typeplant of the same species cultivated under the same conditions.
 12. Atransgenic plant cell, comprising: a gene expression construct, whereinthe construct comprises: i) a nucleic acid molecule comprising asequence having at least 85% identity with SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 45, 47, 49,93, 95, 97, 99, or 101, wherein said nucleic acid molecule isoverexpressed in the transgenic plant cell when compared to plant cellsof the same type in a wild-type plant of the same species; or ii) anucleic acid molecule that encodes a protein comprising a sequencehaving at least 85% identity with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 11, 16, 48, 50, 94,96, 98, 100, or 102, wherein said nucleic acid molecule is overexpressedin the transgenic plant cell when compared to plant cells of the sametype in a wild-type plant of the same species; wherein the sequence isoperatively linked to upstream and downstream regulatory componentscomprising a heterologous promoter that drives the overexpression of thenucleic acid molecule in the plant cell.
 13. The transgenic plant cellof claim 12, comprising: i) a nucleic acid molecule comprising thesequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 45, 47, 49, 93, 95, 97, 99, or 101,wherein said nucleic acid molecule is overexpressed in the transgenicplant cell when compared to plant cells of the same type in a wild-typeplant of the same species; or ii) a nucleic acid molecule that encodes aprotein comprising the sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 94,96, 98, 100, or
 102. 14. The transgenic plant cell of claim 12, whereinthe plant cell comprises a nucleotide sequence having at least 85%identity with SEQ ID NO: 1, 3, 5, 7,
 9. 15. The transgenic plant cell ofclaim 12, wherein the plant cell comprises a nucleotide sequenceencoding a sequence having at least 85% identity with SEQ ID NO:2, 4, 6,8, or
 10. 16. The transgenic plant cell of claim 12, wherein said plantcell is of a monocotyledonous species.
 17. The transgenic plant cell ofclaim 12, wherein said plant cell is of a dicotyledonous species. 18.The transgenic plant cell of claim 12, wherein the plant cell is of agenus selected from the group consisting of: Asparagus, Bromus,Hemerocallis, Hordeum, Lolium, Panicum, Pennisetum, Saccharum, Sorghum,Trigonella, Triticum, Zea, Antirrhinum, Arabidopsis, Arachis, Atropa,Brassica, Browallia, Capsicum, Carthamus, Cichorium, Citrus,Chrysanthemum, Cucumis, Datura, Daucus, Digitalis, Fragaria, Geranium,Glycine, Helianthus, Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Majorana,Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis,Pelargonium, Petunia, Ranunculus, Raphanus, Salpiglossis, Senecio,Sinapis, Solanum, Trifolium, Vigna, and Vitis.
 19. The transgenic plantcell of claim 12, wherein the plant cell is of a species of the genusBrassica.
 20. A transgenic plant, comprising the transgenic plant cellof claim
 12. 21. The transgenic plant of claim 20, wherein plant weight,total weight of leaf or seed, total number of inflorescence, carbonmetabolism, level of carbohydrate, amino acid, lipid production, or seedyield per plant are increased; or bolting time is earlier, when comparedto a wild-type plant of the same species cultivated under the sameconditions.
 22. The method of claim 11, wherein the seed yield per plantis increased by approximately 30% -50% as compared with a wild-typeplant of the same species.
 23. The method of claim 11, wherein thebolting time per plant is earlier by approximately 19% as compared witha wild-type plant of the same species.
 24. The transgenic plant of claim21, wherein the seed yield is increased by approximately 30%-50% ascompared with a wild-type plant of the same species.
 25. The transgenicplant of claim 21, wherein the boiling time is earlier by approximately19% as compared with a wild-type plant of the same species.